Fusion enzyme and application of fusion enzyme in paper-based biosensor

A fusion enzyme, paper-based technology, applied in the field of biological enzyme genetic engineering and biosensing

Active Publication Date: 2021-05-28
BIOLOGY INST OF SHANDONG ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, using the specific affinity adsorption properties of CBM to cellulose to construct fusion enzymes, and using paper base as the immobilizat

Method used

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  • Fusion enzyme and application of fusion enzyme in paper-based biosensor
  • Fusion enzyme and application of fusion enzyme in paper-based biosensor
  • Fusion enzyme and application of fusion enzyme in paper-based biosensor

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Experimental program
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preparation example Construction

[0047] Furthermore, the present invention provides a specific preparation method, that is, transforming the coding gene into Pichia pastoris, and then carrying out the steps of expression and purification.

[0048] The vector used in the present invention is not particularly limited as long as it can replicate in a host cell, and any known vector in the art can be used. Examples of conventional vectors may include natural or recombinant plasmids, cosmids, viruses and phages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon 4A, and Charon 21A can be used as phage vectors or cosmid vectors. As the plasmid vector, pBR type, pUC type, pBluescriptII type, pGEM type, pTZ type, pCL type and pET type can be used. The vector usable in the present invention is not particularly limited, and any known expression vector can be used. Preferably, pDZ, pPIC9k, pACYC184, pCL, pECCG117, pUC57, pBR322, pMW118 or pCC1BAC vectors can be used.

[0049] The second aspect o...

Embodiment 1

[0057] Embodiment 1, fusion expression of CBM and GDH

[0058] Select CBMs sequences with binding specificity to cellulose from CBM1, CBM2, CBM3, CBM5, and CBM10 families, and use structural bioinformatics analysis tools (SWISS-MODEL, ClustalX, VMD, and PyMOl software) to analyze the amino acid frequency and function of different CBMs. Statistical analysis was performed on information such as the framework sequence profile, and CBM2 in Thermobifida fusca was screened out for fusion enzyme construction.

[0059] Based on the analysis of the structural characteristics of CBM2 and GDH, the fusion enzyme molecular linker sequences ((GGGGS)2, (EAAAK)3) were selected from the LinkerDB database, and the endo-β-xylanase (EM_PRO:Z81013. 1) The natural linker sequence connecting the catalytic domain and the CBM2 domain; respectively used as a connecting peptide for the construction of GDH fusion enzyme, the natural linker sequence is "LGGDSSGGGPGEPGGPGGPGEPGGPGGPGEPGGPGDGT", the predict...

Embodiment 2

[0061] Using the pPIC9k-GDH plasmid connected with the codon-optimized Aspergillus niger An76 GDH gene obtained in the previous study as a template, primers were designed to obtain the gene encoding GDH, and the plasmid pUC57-linker-CBM2 DNA was used as a template to design primers to obtain the linker and CBM2 gene. Three kinds of GDH fusion enzyme genes with different connecting peptides were obtained according to the method of Nanjing Nuoweizan homologous recombination kit. The three kinds of GDH fusion enzyme genes were respectively expressed with pPIC9K plasmid and Pichia pastoris GS115 as the expression host to realize GDH fusion enzyme Express.

[0062] (1) GDH fusion enzyme gene cloning:

[0063] Using the principle of homologous recombination and the sequence near the Pichia pastoris GS115 cloning site, six pairs of primers were designed according to the codon-optimized GDH gene sequence and pUC57-linker-CBM2 gene sequence:

[0064]

[0065]

[0066] Using the...

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Abstract

The invention particularly relates to a fusion enzyme and application of the fusion enzyme in a paper-based biosensor. Glucose dehydrogenase (GDH) taking flavin adenine dinucleotide (FAD) as a coenzyme has good stability, is developed into a portable glucose sensor and has a good application prospect. The invention provides a GDH-linker-CBM fusion enzyme constructed by a flavin adenine dinucleotide (FAD) dependent glucose dehydrogenase (GDH) gene and a carbohydrate binding module (CBM) gene, and the GDH-linker-CBM fusion enzyme is prepared into a paper-based sensor through the modification effect of the carbohydrate binding module and a connecting peptide. The enzyme activity, the detection sensitivity and the anti-interference capability of the paper-based sensor are effectively improved, and a good market development prospect is realized.

Description

technical field [0001] The invention belongs to the technical field of bioenzyme genetic engineering and biosensing, in particular to a flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase (GDH) gene and a carbohydrate binding module (CBM) gene constructed The GDH-linker-CBM fusion enzyme and the fusion enzyme are applied in paper-based biosensors. Background technique [0002] The information disclosed in this background section is only intended to increase the understanding of the general background of the present invention, and is not necessarily taken as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to those skilled in the art. [0003] Strict control of physiological blood glucose concentration is the only way for diabetic patients to avoid life-threatening complications, so blood glucose monitoring is an important part of diabetes management. Glycemic control through self-monitoring of blood gluco...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N11/14G01N33/66
CPCC12N9/0006C12Y101/9901C12N11/14G01N33/66C07K2319/20
Inventor 公维丽马耀宏王丙莲刘庆艾郑岚李秋顺韩庆晔蔡雷孟庆军杨艳
Owner BIOLOGY INST OF SHANDONG ACAD OF SCI
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